Interactive Fly, Drosophila

Transcripts are first detected around 7 hours after fertilization, in segmentally repeated clusters in both lateral and medial positions within the ventral somatic mesoderm. There are twice as many clusters in the medial as compared to the lateral positions in each metameric unit. Prior to germ band shortening [Images], new expressing cell clusters appear. As the germ band retracts, three rows emerge along the length of the embryo, one each in dorsal, ventral and pleural regions, corresponding to the locations of the three major embryonic somatic muscles. NAU RNA is not detected in cells of the somatic visceral mesoderm (Michelson, 1990).

Expression of D-mef2, the myocyte specific enhancer factor precedes that of the MyoD homolog nautilus In contrast to nautilus, D-mef2 appears to be expressed in all somatic and visceral muscle cell precursors. Its temporal and spatial expression patterns suggest that D-mef2 may play an important role in commitment of mesoderm to myogenic lineages (Lilly, 1994).

Schneider SL2 cells activate the myogenic program in response to the ectopic expression of daughterless alone, as indicated by exit from the cell cycle, syncytia formation, and the presence of muscle myosin fibrils. Myogenic conversion can be potentiated by the coexpression of Drosophila Mef2 and nautilus with daughterless. In RT-PCR assays Schneider cells express two mesodermal markers, Nautilus and Mef2 mRNAs, as well as very low levels of Daughterless mRNA but no Twist. Full-length RT-PCR products for Nautilus and Mef2 encode immunoprecipitable proteins. RNA-i was used to demonstrate that both endogenous nautilus expression and Mef2 expression are required for the myogenic conversion of Schneider cells by daughterless. Coexpression of twist blocks conversion by daughterless but twist dsRNA has no effect. These results indicate that Schneider cells are of mesodermal origin and that myogenic conversion with ectopic expression of daughterless occurs by raising the levels of Daughterless protein sufficiently to allow the formation of Nautilus/Daughterless heterodimers. The effectiveness of RNA-i is dependent upon protein half-life. Genes encoding proteins with relatively short half-lives (10 h), such as Nautilus or Hsf, are efficiently silenced, whereas more stable proteins, such as cytoplasmic actin or beta-galactosidase, are less amenable to the application of RNA-i. These results support the conclusion that Nautilus is a myogenic factor in Drosophila tissue culture cells with a functional role similar to that of vertebrate MyoD. This is discussed with regard to the in vivo functions of Nautilus (Wei, 2000).

Transheterozygous deficiencies that in combination are reported to remove nautilus do not affect survival nor was somatic muscle formation substantially impacted except in muscles 3 and 19 (Keller, 1998). This is interpreted to suggest that the nautilus gene product is not essential for viability and that nautilus functions only in the formation of a small subset of embryonic muscles. However, several results argue against this interpretation. (1) Injection of nautilus dsRNA into embryos (RNA-i) as well as expression of nautilus antisense RNA in the mesoderm using the Gal4/UAS system both result in a severe loss or absence of muscle in the embryo (Misquitta, 1999); (2) ricin toxin ablation of nautilus-expressing cells completely disrupts the muscle pattern, not just muscles 3 and 19 (Misquitta, 1999); (3) nautilus-expressing cells are incorporated into essentially every somatic muscle in the embryo, as determined by beta-galactosidase expression from the nautilus promoter driving LacZ, and nautilus antibody staining is seen in several newly formed somatic muscles other than muscles 3 and 19, notably muscles 12, 15, 16, 17, 26, and 27 (Wei, 2000).

If the genetic analysis in Drosophila showing in vivo myogenesis without nautilus (Keller, 1998) is correct, this would be the only example of normal muscle development in the complete absence of a MyoD-related protein. The idea is favored that nautilus marks the subset of muscle precursor cells, or founders, that establish the muscle pattern in each hemisegment and that these cells recruit fusion-competent mesodermal cells to complete muscle formation. It is suggested that final activation of the myogenic program requires nautilus expression in every muscle and in vivo and in vitro data support this model. Two recent reports describe the characterization of the immunoglobulin-related genes duf and sns, which are expressed in founder and fusion-competent myoblasts, respectively, during Drosophila myogenesis (Bour, 2000; Ruiz-Gomez, 2000): both genes are essential for myoblast fusion. It is not clear if nautilus expression is restricted to duf-positive myoblasts but the results presented here would predict this to be the case, based upon the lack of muscle development in embryos ablated for the nautilus-expressing cells. It is not known if duf or sns are also expressed in myogenically converted Schneider cells but this will be examined further (Wei, 2000).

Effects of Mutation or Deletion

In the Drosophila embryo, nautilus is expressed in a subset of muscle precursors and differentiated fibers and is capable of inducing muscle-specific transcription, as well as myogenic transformation. In this study, an examination was made of the consequences of nautilus loss-of-function on the development of the somatic musculature. Genetic and molecular characterization of two overlapping deficiencies, Df(3R)nau-9 and Df(3R)nau-11a4, reveal that both of these deficiencies remove the nautilus gene without affecting a common lethal complementation group. Individuals transheterozygous for these deficiencies survive to adulthood, indicating that nautilus is not an essential gene. These embryos are, however, missing a subset of muscle fibers, that includes the dorsal oblique, dorsal acute and lateral longitudinals, providing evidence that (1) some muscle loss can be tolerated throughout larval development and (2) nautilus does play a role in muscle development. In addition to the absence of particular muscle fibers in transheterozygotes, novel muscle fibers are occasionally present in these embryos. The appearance of these fibers provides support for the hypothesis that, in the absence of nau, precursors to muscles such as 3 and 19 can undergo further myogenic differentiation. In some cases, these novel fibers have features reminiscent of specific muscles. For example, a novel muscle fiber is seen in a position and orientation similar to that of muscle 2. It is enticing to consider the possibility that this muscle arises from a precursor to muscle 3 and its differentiation program has been diverted by the lack of Nau expression. Examination of muscle precursors in these embryos reveals that nautilus is not required for the formation of muscle precursors, but rather plays a role in their differentiation into mature muscle fibers. It is suggested that nautilus functions in a subset of muscle precursors to implement their specific differentiation programs (Keller, 1998).

The expression of the MyoD gene homolog, nautilus (nau), in the Drosophila embryo defines a subset of mesodermal cells known as the muscle 'pioneer' or 'founder' cells. These cells are thought to establish the future muscle pattern in each hemisegment. Founders appear to recruit fusion-competent mesodermal cells to establish a particular muscle fiber type. In support of this concept every somatic muscle in the embryo is associated with one or more nautilus-positive cells. However, because of the lack of known (isolated) nautilus mutations, no direct test of the founder cell hypothesis has been possible. Toxin ablation and genetic interference by double-stranded RNA (RNA interference or RNA-i) have been used to determine both the role of the nautilus-expressing cells and the nautilus gene, respectively, in embryonic muscle formation. In the absence of nautilus-expressing cells muscle formation is severely disrupted or absent. A similar phenotype is observed with the elimination of the nautilus gene product by genetic interference upon injection of nautilus double-stranded RNA (Misquitta, 1999).

To test whether the direct ablation of nautilus mRNA would result in a disrupted muscle phenotype in the Drosophila embryo, the gal4/UAS system was used to express nautilus antisense RNA throughout the mesoderm. Females from the gal4 enhancer trap line 24B containing the twi-gal4 transgene were crossed with males from four independent lines homozygous for a gal4 UAS antisense nautilus transgene containing only the coding region in reverse orientation. The degree of disruption in the muscle pattern of the progeny flies depends partially on the particular antisense transgenic line used in the cross. Previous studies with the overexpression of nautilus gave a phenotype that includs the formation of some additional muscles and a disruption of the heart tube, presumably because of the formation of skeletal muscle cells in the heart tube itself. With the additional results from the antisense induction experiments it is concluded that nautilus plays a major role in the formation of the muscle pattern and may be involved in the determination of the muscle founder cell lineage in the embryo, because the muscle phenotypes resulting from the ricin ablation of the nautilus-positive cells and the nautilus antisense expression are so similar. These results define a crucial role for nautilus in embryonic muscle formation. The application of RNA interference to a variety of known Drosophila mutations as controls gives phenotypes essentially indistinguishable from the original mutation. RNA-i provides a powerful approach for the targeted disruption of a given genetic function in Drosophila (Misquitta, 1999).

The results from the injection of nautilus dsRNA point to a more general approach for the analysis of gene function during Drosophila development and suggest that the RNA interference method essentially would mimic a gene knock-out in the injected generation of Drosophila embryos. To test this idea a variety of cDNA clones were obtained representing a maternal gene expressed in the embryo (daughterless); additional genes involved in myogenesis (S59, DMEF2); homeobox genes (engrailed and S59); a gene important for gastrulation (twist), and a gene expressed in the adult eye (white). This panel of genes covers most stages of Drosophila development. twist was initiatially tested because the mutant has a clear phenotype that is easy to score when compared with wild-type larva. The injection of twist dsRNA (the complete coding region) into embryos produces a twisted larval phenotype that is indistinguishable from the original twist mutation. Similarly, injection of the first 1,200 bp of engrailed dsRNA produces the compressed dentical belt pattern characteristic of an engrailed null mutant. Daughterless mRNA is both maternally loaded and expressed zygotically, and the mutant phenotype produces very characteristic disruptions in the central nervous system (CNS) and peripheral nervous system (PNS). It has been shown previously that mex3, a maternally loaded RNA in C. elegans, can be ablated by dsRNA injection into the gonads. daughterless dsRNA (complete coding region) was injected and the characteristic neuronal phenotypes were sought by using the mAb MAB 22C10. The CNS as well as the PNS were disrupted to varying degrees in the injected embryos. The severity of the phenotype consistently shows a CNS disruption with a variable PNS pattern, possibly reflecting the fact that the CNS is formed before the PNS. This result suggests that maternally loaded as well as zygotically expressed RNA can be affected by RNA-i in Drosophila. The homeobox gene S59 marks a subset of muscle founder cells for 5 of 29 muscles in each hemisegment of the embryo corresponding to muscles 5, 18, 25, 26, and 27. Embryos with an S59 lacZ transgene marking muscles 18 and 25 were injected with S59 dsRNA (complete coding region). In this case, the S59-specific lacZ antibody-staining pattern is abolished. The total muscle pattern for embryos injected with S59 dsRNA, although disrupted, still shows the presence of poorly organized muscle groups in each hemisegment. This is unlike the almost complete absence of muscle observed with the injection of nautilus dsRNA. DMEF2, a member of the MADS domain transcription factor family, is essential for muscle formation in Drosophila. The DMEF2 / embryo has no muscle and is missing the characteristic gut constrictions found in the uninjected embryo. Injection of DMEF2 dsRNA (complete coding region) results in embryos that lack any detectable muscle and an absence of gut morphology (Misquitta, 1999).

Because particular RNA interference phenotypes are transferable to the next generation of C. elegans, it was particularly interesting to see whether genes expressed in the adult eye could be affected by the injection of dsRNA into the embryo. The white gene was chosen, even though it is expressed throughout embryogenesis: it was asked if any aspect of the white-eyed mutant phenotype could be observed after the injection of white dsRNA (the first 500 bp from the P element minigene) into wild-type embryos with red eyes. Phenotypes indicating interference with white gene function were observed in response to the RNA interference with white dsRNA, although the frequency of the mutant phenotype is extremely low (<3%) when compared with the level of typical mutant phenotypes scored in the embryos injected with dsRNA (>75%). Similar to the results reported in C. elegans, very few molecules of white dsRNA appear to be required to obtain some evidence of interference with white gene function in the adult eye, because on the order of only107 molecules were injected. This last result supports the idea that RNA interference is acting catalytically because the transition from embryo to adult fly would substantially dilute the injected dsRNA (Misquitta, 1999).

nautilus (nau), the single Drosophila member of the bHLH-containing myogenic regulatory family of genes, is expressed in a subset of muscle precursors and differentiated fibers. It is capable of inducing muscle-specific transcription as well as myogenic transformation, and plays a role in the differentiation of a subset of muscle precursors into mature muscle fibers. The nau zygotic loss-of-function phenotype has been determined using genetic deficiencies in which the gene is deleted. This genetic loss-of-function phenotype differs from the loss-of-function phenotype determined using RNA interference (Misquitta, 1999). The present study re-examines this loss-of-function phenotype using EMS-induced mutations that specifically alter the nau gene, and extends the genetic analysis to include the loss of both maternal and zygotic nau function. In brief, embryos lacking nau both maternally and zygotically are missing a distinct subset of muscle fibers, consistent with its apparent expression in a subset of muscle fibers. The muscle loss is tolerated, however, such that the loss of nau both maternally and zygotically does not result in lethality at any stage of development (Balagopalan, 2001).

The subtle muscle phenotype exhibited by the deficiency embryos represents the zygotic loss-of-function phenotype. Although zygotic expression of nau is not essential for survival to adulthood, the eggs of adult females lacking nau exhibit severely reduced rates of fertilization. While a clear explanation for this mutant phenotype remains to be determined, it has facilitated the isolation of EMS-induced nau specific mutations termed nau. Embryos with homozygous null mutations are missing a subset of muscle fibers. Individuals lacking these muscles still survive to adulthood. Analysis of RNA from ovaries and unfertilized eggs did not reveal maternally provided nau transcripts that might account for the difference between the zygotic loss-of-function phenotype and that observed with RNAi (Misquitta, 1999). Interpretation of these results as conclusive was prevented, however, by the potential for alternative forms of the nau mRNA that may not be detected by the chosen oligonucleotides. In addition, RT-PCR analyses could not address whether nau protein was provided to the egg. These explanations for the subtle zygotic loss-of-function phenotype could be eliminated only by the generation of null alleles and subsequent analysis of germline clone embryos. These embryos were derived from ovaries in which the germline cells, and resulting eggs, were lacking nau genetically and were fertilized by nau mutant sperm. The elimination of any hypothetical maternally-provided nau does not alter the loss-of-function phenotype or survival rate of embryos lacking nau. These observations therefore do not support the existence of a maternal contribution for nau, confirming that nau is essential for the formation of only a subset of muscle fibers but not adult viability (Balagopalan, 2001).

The subtle muscle phenotype observed in flies lacking nau is in contrast to the more critical role that the vertebrate Myogenic Regulatory Factors (MRFs) play in vertebrate myogenesis, and was not anticipated at the time of its initial isolation. Such early expectations might, however, be somewhat naive in the context of current understanding of Drosophila myogenesis. Specification of the elaborate pattern of larval body wall muscles actually begins concurrent with the earliest stages of myogenesis in the Drosophila embryo. Distinct equivalence groups composed primarily of post-mitotic myoblasts segregate from the mesoderm at specific locations. In a process of lateral inhibition mediated by Notch, a single muscle progenitor will then be selected from the cells within each equivalence group. This single founder cell, which may undergo one additional mitotic division, then seeds the fusion process and controls the unique features of the resulting muscle fiber. Thus, the larval body wall muscles that develop in a Drosophila embryo are not derived from a common pool of homogeneous myoblasts, and appear to segregate from the mesoderm with distinct features. The results presented here establish that nau, the single Drosophila homolog of the MRFs, is not required for determination of all embryonic myoblasts. Indeed, no factor has yet been identified that is specifically required for the determination of all myoblasts. In fact, based upon its pattern of expression and ectopic behavior, Drosophila Twist may serve such a function, making a general role for nau unnecessary. Alternatively, one might anticipate the existence of several factors that, through individual and combinatorial mechanisms, are responsible for differentiation of founder myoblasts. Consistent with this prediction, several genes are expressed in subsets of founder myoblasts and, in at least some cases, are essential for the development of a subset of muscle fibers. It seems plausible that nau is simply another example of a gene that serves such a function (Balagopalan, 2001).

It is noted that the roles of other highly conserved proteins in Drosophila myogenesis are in some contrast to their roles in vertebrate myogenesis. For example, murine TWI is a powerful negative regulator of skeletal muscle differentiation, whereas a high level of Drosophila Twi is a critical determinant of somatic myogenesis. In addition, although the C. elegans twi homolog hlh-8 is involved in the development of a subset of non-striated muscle, it is not required for formation of striated muscle. Definitive comparisons of the functions of the vertebrate and invertebrate MEF2 family members in skeletal and somatic myogenesis remain incomplete or are precluded by early lethality. However, even invertebrate family members play distinctly different roles. For example, embryos lacking the single Drosophila family member Dmef2 exhibit severe defects in the differentiation of all three muscle lineages: somatic, cardiac, and visceral, while the single C. elegans MEF2 homolog is not essential for myogenesis. Finally, distinct differences in function have been observed among members of the MRF family of bHLH proteins. In contrast to the critical role in all muscle fibers revealed by mouse knockouts for the MRFs, these data suggest a nonessential muscle fiber-specific role for nau. Yet another role seems to be suggested by the analysis of mutations in CeMyoD, the single C. elegans MRF homolog. In brief, CeMyoD appears to be essential for the integrity of the muscle fibers but is not necessary for their formation (Balagopalan, 2001).

Combinatorial coding of Drosophila muscle shape by Collier and Nautilus

The diversity of Drosophila muscles correlates with the expression of combinations of identity transcription factors (iTFs) in muscle progenitors (see Restricted gene expression patterns in somatic muscles). This study addresses the question of when and how a combinatorial code is translated into muscle specific properties, by studying the roles of the Collier and Nautilus iTFs that are expressed in partly overlapping subsets of muscle progenitors. The three dorso-lateral (DL) progenitors which express Nautilus and Collier are specified in a fixed temporal sequence, and each expresses additionally other, distinct iTFs. Removal of Collier leads to changes in expression of some of these iTFs and mis-orientation of several DL muscles, including the dorsal acute DA3 muscle which adopts a DA2 morphology. Detailed analysis of this transformation revealed the existence of two steps in the attachment of elongating muscles to specific tendon cells: transient attachment to alternate tendon cells, followed by a resolution step selecting the final sites. The multiple cases of triangular-shaped muscles observed in col mutant embryos indicate that transient binding of elongating muscle to exploratory sites could be a general feature of the developing musculature. In nau mutants, the DA3 muscle randomly adopts the attachment sites of the DA3 or DO5 muscles that derive from the same progenitor, resulting in a DA3, DO5-like or bifid DA3-DO5 orientation. In addition, nau mutant embryos display thinner muscle fibres. Together, these data show that the sequence of expression and combinatorial activities of Col and Nau control the pattern and morphology of DL muscles (Enriquez, 2012).

The larval Drosophila somatic musculature is made of a stereotyped set of about 30 uniquely identifiable muscles per hemisegment. This study shows that the combinatorial activities of Col and Nau are required to establish the pattern of DL muscles and confer upon these muscles their distinctive shapes and epidermal attachment sites (Enriquez, 2012).

Col is expressed in a promuscular cluster and the three derived PCs at the origin of DL muscles. Each of these PCs is specified at a stereotypic position and according to a precise temporal sequence, with the dorsal DA3/DO5 PC being born first and the ventral LL1/DO4 PC being born last. In addition to Col and Nau, each expresses a combination of specific iTFs, including Kr, Poxm and S59. Expression of a specific set of iTFs in each DL PC could thus integrate both positional and temporal cues. The textbook view is that, similar to neuroblast selection in the neuroectoderm, each muscle PC is selected via the process of lateral inhibition from an equivalence group of mesodermal cells. The parallel between neuroblast and PC selection is supported by the co-expression of the proneural gene l(1)sc and iTFs such as Eve or S59 in specific promuscular clusters. However, a deficiency of l(1)sc results in only minor defects of somatic muscle development and does not prevent the selection of the DA3/DO5 and DT1/DO3 progenitors. A possibility is that several PCs could be selected from large competence domains defined by expression of specific iTFs and l(1)sc clusters play only a limited or redundant role. Selection of the three DL PCs from a cluster of Col-expressing cells supports this view. The fact that the DA3/DO5 and LL1/DO4 PCs are born sequentially and positioned adjacent to one another, suggests a reiterative selection process (Enriquez, 2012).

The most obvious muscle pattern defects that are observed in col mutant embryos, are DA3 > DA2 and DA3 > LL1 transformations. Since the DA2, DA3 and LL1 muscles are derived from different PCs, these defects indicate changes in progenitor identity. In one case, Poxm and S59 expression in the DT1/DO3 progenitor requires Col activity. In contrast, Kr expression in the LL1/DO4 PC is independent of Col. Interestingly, Kr and S59 are expressed together in the ventral VA1/VA2 PC but, in this case, Kr regulates S59 expression. Together, these expression data strengthen the concept of combinatorial coding of muscle identity at the PC stage and show that hierarchies of interactions between different iTFs are progenitor-specific (Enriquez, 2012).

In Poxm mutants, the DO3 muscle is often duplicated, likely at the expense of a DT1 muscle that is often missing. In S59 mutant embryos, the DO3 and DT1 sibling muscles share ventral attachment sites and form a single syncitium in a fraction of segments. Since Col acts upstream of Poxm and S59 in the DT1/DO3 progenitor, it was expected the col mutant phenotype to overlap with the poxm and S59 phenotypes. It may not be so simple, however, since the DT1 muscle is absent only in a small fraction of segments in col mutant embryos. Interestingly, while the DA3 muscle is transformed into a DA2-like muscle in absence of Col activity, the LL1 muscle can adopt a DA3 morphology The LL1 muscle is mis-oriented in col as well as in Kr mutant embryos. Together, these muscle re-orientation phenotypes suggest that there is a range of possible attachment sites for each elongating DL muscle and that the final pattern results from a global combinatorial control. The propensity of elongating muscles to explore several attachment sites, could explain why a coordinate, global regulation by combinations of iTFs is essential. The term regulatory state has been used to describe the total set of active transcription factors in a given cell at a given time. In essence, each PC iTF code is an example of a regulatory state. The loss of one iTF reveals an alternative regulatory state and PC identity, suggesting that a given iTF is able to exert its activity only in the presence of other specific iTFs. A global analysis of this mutual dependency now requires the identification of all DL iTFs, including those expressed in the DO3, DO4 or DO5 muscles (Enriquez, 2012).

Nau differs from other well characterised iTFs, in that it is expressed in most, if not all FCs, before being restricted to specific muscle precursors. SEM analysis shows that most muscles are much thinner in nau mutant than wt embryos. Detailed examination of the mutant DA3 muscle showed that, despite being thinner, it contained a number of nuclei close to normal. nau activity is thus required for embryonic muscle fibre size, but not the muscle fusion programme, per se. Whether Nau directly or indirectly regulates the synthesis and/or assembly of myofibril proteins remains to be determined. DL muscles, including the DA3 muscle, are more severely affected. Taken together, these data lead to the conclusion that Nau performs both general myogenic functions and specific functions in selected muscle lineages. A different threshold level of MRF activity might be needed to initiate myogenesis in different trunk and craniofacial muscles. The different Nau functions in establishing the Drosophila muscle pattern suggest that Nau activity is, in part conditioned by interactions with other iTFs such as Col (Enriquez, 2012).

Co-expression of Nau and Col in the DA3/DO5 progenitor provides a good model to challenge the concept of combinatorial control of muscle identity. While transformed towards a DA2 muscle in absence of Col activity, the DA3 muscle adopts the morphology of its sibling, DO5 muscle in absence of Nau. Thus, while co-expressed in the DA3/DO5 PC, Col and Nau act at different steps in the DA3 lineage. The following regulatory cascade is proposed: Col expression in a large cluster of myoblasts and the three derived PCs, under control of an early CRM and Hox activity, defines a domain of competence for DL muscle development. Col activity, either upstream, and/or in parallel to other iTFs, contributes to confer each DL progenitor its particular identity. The restricted ability of Col in maintaining its own expression in the DA3 FC, by direct binding to a late, DA3-specific CRM, reveals a context-dependence provided by the iTF combination specific to the DA3/DO5 PC. This PC-specific handover process may explain why the DA3 muscle is the most frequently affected in col mutant embryos. Asymmetric division of each DL PC generates two FCs with different regulatory states. Whereas two DA3 and two DO5 muscles form in Notch (N) loss- and gain of function conditions, respectively, Nau confers robustness to the DA3 versus DO5 differentiation programme. This Nau function involves positive regulation of col transcription in the DA3 syncytium nuclei and is independent of Nau function in ensuring normal fibre size (Enriquez, 2012).

In conclusion, these data show that the sequence of expression and combinatorial activities of Col and Nau are required to establish the pattern of DL muscles and confer upon the DA3 muscle its distinctive size and epidermal attachment sites. Identification of the gene targets of this combination is now essential to link a sequence of regulatory states to the architecture of a specific Drosophila muscle. Interestingly, a recent report suggests that EBF cooperates with MyoD in driving aspects of differentiation in Xenopus muscle cells, suggesting that there may be an ancient, evolutionarily conserved, transcriptional relationship between the COE/EBF and MyoD gene families (Enriquez, 2012).

Embryonic muscles connect to the chitinous exoskeleton of the developing embryo via tendon cells, which are specialised epidermal cells. Proper attachment of muscles requires the specific targeting of tendon cells at segmental or intra-segmental, stereotypic positions. The general view is that growing myotubes extend filopodia at their two ends, in search of attachment sites, and that muscle extension ceases when muscles have reached their targeted tendon cells. Some muscle guidance components have been described, such as the Derailed receptor tyrosine kinase for the lateral transverse muscles and the Robo and Robo2 receptors, the transmembrane protein Kon-Tiki and its associated intracellular signalling protein dGrip for ventral-longitudinal muscles. How the precise matching of specific muscles to specific tendon cells is achieved, however, is far from being understood. SEM analysis and phalloidin staining of col mutant embryos showed many mis-oriented muscles, suggesting targeting defects. Many fibres showed more than two attachment sites to the epidermis, however, a phenotype difficult to reconcile with a bipolar extension of muscle precursors until they connect to the epidermis. Rather, the observation that the wt DA3 muscle is transiently attached to three sites, before acquiring its fully extended bipolar morphology, indicates the existence of an exploratory step, followed by a resolution step that selects the final attachments sites. The allelic series of col phenotypes, which revealed many triangular shape fibres, indicates a defect in the resolution process, without ruling out that ventral elongation of the DA3 myofibre is also defective. Terminal differentiation of tendon cells is dependent upon their interaction with muscles and tendon cells could play a role in the resolution step. Triangular shape LO1 muscles were previously observed in mutants for dgit, which encodes a GTPase activator protein that is involved in myotube guidance. Based on the dgit phenotype, and the current observations, it is proposed that the migratory path of muscles towards their targeted tendon cells can involve exploratory attachment to tendon cells along this path. Deciphering how the final, stereotyped, pattern is controlled now requires the identification of how various iTF combinations differentially regulate guidance cues (Enriquez, 2012).

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